Protein Expression and Purification
The modified pPEP-T vector [39] was kindly provided by the M. E. Müller Institute, Basel, Switzerland. The genes encoding P6c and P11c protein were placed between NcoI and EcoRI restriction sites. The plasmids were then transformed into the Escherichia Coli strain BL21(DE3)pLysS expression cells (Novagen, Madison, WI, USA). The bacteria were incubated at 37°C in Luria Broth (LB) medium in the presence of 200 mg/ml ampicillin and 30 mg/ml chloramphenicol. Expression was induced by adding 1 mM isopropyl β-D-thiogalactopyranoside. After 3 hours of expression, the bacteria were collected by centrifugation at 4000 g for 15 min. The bacterial pellet was resuspended and lysed in a lysis buffer (9 M urea, 100 mM NaH2PO4, 10 mM Tris, 10 mM β-mercaptoethanol, and pH 8.0) by sonication. The cell debris was removed by centrifugation at 305000 g for 45 min. The supernatant was then incubated with Ni-NTA Agarose beads (Qiagen, Valencia, CA, USA) overnight and then loaded into a column. The protein contaminants were removed by washing the column sequentially with pH buffers 6.3, 5.9 and 5.0, which contain 9 M urea, 100 mM NaH2PO4, 20 mM sodium citrate, 10 mM imidazole and 10 mM β-mercaptoethanol. The P6c proteins were then eluted by the elution buffer containing 9 M urea, 100 mM NaH2PO4, 10 mM Tris, 500 mM imidazole, 10 mM β-mercaptoethanol and pH 8.0. The purity of the P6c proteins was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis.
Protein refolding procedure
The P6c protein was first denatured in a urea-containing buffer (9 M urea, 20 mM HEPES, 150 mM NaCl, 5% Glycerol, pH 7.5), and then concentrated to 1 mg/ml. The protein was refolded by adding it drop wise to the refolding buffer (20 mM HEPES, 150 mM NaCl, 5% Glycerol, pH 7.5), until the protein concentration reached a concentration of 0.05 mg/ml. The samples were then dialyzed overnight in the refolding buffer to remove the remaining urea.
GNPs
The citrated-coated gold nanoparticles of the three gold core sizes of 5, 10 and 15 nm, respectively, were purchased from Nanocs Inc., New York, USA. The stock concentrations of the 5, 10 and 15 nm citrate-coated gold nanoparticles were approximately 0.083, 0.0095 and 0.0023 nmole/ml, respectively.
The PEG-coated gold nanoparticles with two gold core sizes of 5 and 10 nm were purchased from Nanocs Inc., New York, USA. The stock concentrations of the 5 and 10 nm PEG-coated gold nanoparticles were approximately 0.083 and 0.0095 nmole/ml, respectively.
The polymer-coated gold nanoparticles with carboxyl or amine surface functional groups were purchased from Ocean NanoTech Inc., AR, USA. Both polymer-coated gold nanoparticle had 5 nm gold cores. The gold nanoparticles with carboxylic acid groups were coated with dodecanethiol and a monolayer of amphiphilic polymer. The zeta potential of these gold nanoparticles is −30 mV to −50 mV (provided by the supplier). The concentration of the gold nanoparticles with carboxylic acid groups was about 5 mg/ml, which gives a concentration of approximately 6.7 nmole/ml. The gold nanoparticles with amine groups were coated with amphiphilic polymer and PEG. The zeta potential of these gold nanoparticles is −10 mV to +10 mV (provided by the supplier). The concentration of the gold nanoparticles with amine groups was about 1 mg/ml, which gives a concentration of approximately 1.3 nmole/ml.
Encapsulation of citrate-coated gold nanoparticles by P6c SAPNs
The P6c proteins were first denatured in the denaturing buffer (9 M urea, 20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol). Then the denatured proteins were concentrated to about 1 mg/ml using the Amicon centrifuge filter (5000 MWCO, Millipore, MA, USA).
The 5 nm citrate-coated nanoparticles were diluted in the refolding buffer to a concentration of approximately 0.0047 nmole/ml. Three different refolding buffers were used for dilution of gold nanoparticles. The refolding buffers were composed of 20 mM HEPES pH 7.5, 5% glycerol, and 10, 75, and 150 mM NaCl, respectively. Then, the denatured P6c protein solution (~1 mg/ml) was added drop wise to the GNP-refolding buffer until the protein concentration reached a concentration of 0.05 mg/ml (3.96 nmole/ml) in the final protein-GNP solution. The protein-GNP solution was then dialyzed overnight against the buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol) to remove the remaining urea.
The encapsulation procedures for the 10 nm citrate-coated gold nanoparticles were similar to that for the 5 nm citrate-coated gold nanoparticles. The 10 nm citrate-coated gold nanoparticles were first diluted in the refolding buffer containing 10 mM HEPES pH 7.5, 75 mM NaCl, and 5% glycerol. Then, the denatured P6c protein solution (~1 mg/ml) was added drop wise to the GNP-refolding buffer until the protein concentration reached a concentration of 0.05 mg/ml (3.96 nmole/ml) in the final protein-GNP solution. The protein-GNP solution was then dialyzed overnight against the buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol) to remove the remaining urea. Three different molar ratios of gold nanoparticles to proteins were used for the encapsulation: (a) The P6c protein concentration was 0.05 mg/ml (approximately 4 nmol/ml). The 10 nm GNPs concentration was 4.7×10-4 nmol/ml. (b) The P6c protein concentration was 0.05 mg/ml. The 10 nm GNPs concentration was 4.7×10-3 nmol/ml. (c) The P6c protein concentration was 0.025 mg/ml. The 10 nm GNPs concentration was 4.7×10-3 nmol/ml.
The encapsulation procedures for the 15 nm citrate-coated gold nanoparticles were also similar to that for the 5 nm citrate-coated gold nanoparticles. The 15 nm citrate-coated gold nanoparticles were first dissolved in the refolding buffer containing 10 mM HEPES pH 7.5, 75 mM NaCl, and 5% glycerol. Then, the denatured P6c protein solution (~1 mg/ml) was added drop wise to the GNP-refolding buffer, until the protein concentration reached a concentration of 0.05 mg/ml (3.96 nmole/ml) in the final protein-GNP solution. The protein-GNP solution was then dialyzed overnight against the buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol) to remove the remaining urea. Two different molar ratios of gold nanoparticles to proteins were used for the encapsulation: (a) The P6c protein concentration was 0.05 mg/ml (approximately 4 nmol/ml). The 10 nm GNPs concentration was approximately 2.3×10-3 nmol/ml. (b) The P6c protein concentration was 0.025 mg/ml. The 10 nm GNPs concentration was approximately 2.3×10-3 nmol/ml.
Encapsulation of PEG-coated gold nanoparticles by P6c SAPNs
The PEG-coated gold nanoparticles were first diluted in the refolding buffer containing 10 mM HEPES pH 7.5, 75 mM NaCl, and 5% glycerol to a concentration of approximately 0.0047 nmole/ml. Then, the denatured P6c protein solution (~1 mg/ml) was added drop wise to the GNP-refolding buffer until the protein concentration reached 0.05 mg/ml (3.96 nmole/ml) in the final protein-GNP solution. The protein-GNP solution was then dialyzed overnight against the buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol) to remove the remaining urea.
Encapsulation of polymer-coated gold nanoparticles with carboxyl or amine surface functional groups by P6c SAPNs
The polymer-coated gold nanoparticles were diluted to approximately 0.01 nmole/ml in the refolding buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol). Then, the denatured P6c protein solution (~1 mg/ml) was added drop wise to the GNP-refolding buffer, until the protein concentration reached a concentration of 0.05 mg/ml (3.96 nmole/ml) in the final protein-GNP solution. The protein-GNP solution was then dialyzed overnight against the buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol) to remove the remaining urea.
Encapsulation of gold nanoparticles by P11c SAPNs
The encapsulation procedures for gold nanoparticles by the P11c SAPNs were similar to the procedures for the P6c SAPNs. P11c SAPNs were used for the encapsulation of all three kinds of gold nanoparticles mentioned above. The concentration of the P11c proteins was kept as 0.05 mg/ml for all the encapsulation samples. In the final encapsulation samples, approximately 0.0047 nmole/ml of the citrate-coated gold nanoparticles were used. The concentrations of the polymer-coated gold nanoparticles were also approximately 0.01 nmole/ml in their encapsulation samples.
Dynamic light scattering
The hydrodynamic diameter was determined with a Malvern Zetasizer Nano S equipped with a 633 nm laser. Hellma Quartz cuvettes with a 3 mm light path and centre 9.65 mm were used (Cat. No. 105.251.005-QS). The measurements were performed at 20°C using 80 μl samples. All the samples were filtered once using 0.1 μm Millex-VV filter (Millipore, MA, USA) before measurement. The volume-average hydrodynamic sizes were reported by the Malvern DTS software, version 6.01.
Transmission Electron Microscopy
A drop of 5 μl sample was placed on a 400 mesh copper grid coated with Formvar/carbon film (Electron Microscopy Sciences, PA, USA) for 1 min. The grid was washed sequentially by three drops of 5 μl distilled water. Then the sample was negatively stained with a drop of 5 μl 1% uranyl acetate (SPI Supplies, PA, USA) for 1 min. Excess stain solution was removed by Whatman filter paper, before the grid was slowly dried at room temperature. Electron micrographs were taken with an FEI Tecnai T12 transmission electron microscope at an accelerating voltage of 80 kV.
The TEM images were first inspected with Photoshop CS4 (Adobe, San Jose, CA). The particles were selected and filled manually using the selection tools in Photoshop CS4, omitting the very small particles or background (area less than 50 nm2) and large aggregates (area larger than 5000 nm2). Image analysis was then performed with the public domain software ImageJ [40]. Then, the Feret diameter obtained by ImageJ was used to describe the size of the particles.